Conformation-activity relationship of apoptosis-inducing phosphodiester oligonucleotides

ABSTRACT

The present invention facilitates in silico evaluation of molecules for biological activity by providing a computer-based method to predict whether oligonucleotides may possess biological activity and the efficacy of the biological activity based on the three-dimensional structure and charge characteristics of the oligonucleotides. Biological activities include, but are not limited to, cellular proliferation, induction of cell cycle arrest and apoptosis.

PRIOR RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisionalpatent application serial No. 60/313,290 filed Aug. 17, 2001.

FIELD OF THE INVENTION

[0002] The present invention provides a computer-based method to predictwhether oligonucleotides may induce apoptosis in cancer cells based onthe three-dimensional structure and charge characteristics of theoligonucleotides.

BACKGROUND OF THE INVENTION

[0003] Cancer is an aberrant net accumulation of atypical cells, whichcan result from an excess of proliferation, an insufficiency of celldeath, or a combination of the two.

[0004] Proliferation is the culmination of a cell's progression throughthe cell cycle resulting in the division of one cell into two cells. Thefive major phases of the cell cycle are G₀, G₁, S, G₂, and M. During theG₀, phase, cells are quiescent. Most cells in the body, at one time, arein this stage. During the G₁phase, cells, responding to signals todivide, produce the RNA and the proteins necessary for DNA synthesis.During the S-phase (SE, early S-phase; SM, middle S-phase; and SL, lateS-phase) the cells replicate their DNA. During the G₂ phase, proteinsare elaborated in preparation for cell division. During the mitotic (M)phase, the cell divides into two daughter cells. Alterations in cellcycle progression occur in all cancers and may result fromover-expression of genes, mutation of regulatory genes, or abrogation ofDNA damage checkpoints (Hochhauser D., Anti-Cancer ChemotherapeuticAgents, 8:903, 1997).

[0005] Apoptosis or programmed cell death is the physiological processfor the killing and removal of unwanted cells and the mechanism wherebychemotherapeutic agents kill cancer cells. Apoptosis is characterized bydistinctive morphological changes within cells that include condensationof nuclear chromatin, cell shrinkage, nuclear disintegration, plasmamembrane blebbing, and the formation of membrane-bound apoptotic bodies(Wyllie et al., Int. Rev. Cytol., 68: 251, 1980). The translocation ofphosphatidylserine from the inner face of the plasma membrane to theouter face coincides with chromatin condensation and is regarded as acellular hallmark of apoptosis (Koopman, G. et al., Blood, 84:1415,1994). The actual mechanism of apoptosis is known to be mediated by theactivation of a family of cysteine proteases, known as caspases.However, most prior art anti-cancer therapies are directed to inductionof apoptosis, have proven to be less than adequate for clinicalapplications. Many of these therapies are inefficient or toxic, haveadverse side effects, result in development of drug resistance orimmunosensitization, and are debilitating for the recipient. Manydiseases or conditions are characterized by undesired cellularproliferation and are know to one of ordinary skill in the medical orveterinary arts.

[0006] Induction of programmed cell death via the induction ofsenescence (Dimri et al., Proc. Natl. Acad. Sci USA 92:20, 1995) orapoptosis (Wyllie et al., Int. Rev. Cytol. 68:251, 1980) is importantfor the treatment of disorders that involve aberrant accumulation ofunwanted cells such as, but not limited to, cancer, autoreactive,autoimmune, inflammatory and proliferative disorders. However, mostprior art anti-cancer therapies, whether directed to induction ofapoptosis or to stimulation of the immune system, have proven to be lessthan adequate for clinical applications. Many of these therapies areinefficient or toxic, have adverse side effects, result in developmentof drug resistance or immunosensitization, and are debilitating for therecipient. New methods are needed for evaluating molecules to predictwhether they will possess a desired biological activity.

[0007] Synthetic oligonucleotides are polyanionic sequences that areinternalized in cells (Vlassov et al., Biochim. Biophys. Acta, 11197:95,1994). Synthetic oligonucleotides are reported that bind selectively tonucleic acids (Wagner, R., Nature, 372:333, 1994), to specific cellularproteins (Bates et al., J. Biol. Chem., 274:26369, 1999) and to specificnuclear proteins (Scaggiante et al., Eur. J. Biochem, 252:207, 1998) inorder to inhibit proliferation of cancer cells.

[0008] Synthetic 27 base sequences containing guanine (G) and variableamounts of thymine (T) (oligonucleotides GTn) wherein n is ≧1 or ≦7 andwherein the number of bases is ≧20 (Scaggiante et al., Eur. J. Biochem.,252:207, 1998), are reported to inhibit growth of cancer cell lines bysequence specific binding to a 45 kDa nuclear protein, whereas GTn,wherein the number of bases is ≦20, are reported to be inactive againstcancer cell lines (Morassutti et al., Nucleosides and Nucleotides,18:1711, 1999). Two synthetic GT-rich oligonucleotides of 15 and 29bases with 3′ aminoalkyl modifications are reported to form G-quartetsthat bind to nucleolin and to inhibit proliferation of cancer cell lines(Bates et al., J. Biol. Chem., 274:26369, 1999). The synthetic six baseTTAGGG-phosphorothioate, having a sequence identical to that of themammalian telomere repeat sequence, is reported to inhibit proliferationof Burkitt's lymphoma cells in vitro and in vivo (Mata et al., Toxicol.Applied Pharmacol., 144:189, 1997). However, the synthetic six baseTTAGGG-phosphodiester nucleotide is reported to have no anti-telomeraseactivity (U.S. Pat. No. 5,643,890).

[0009] Deoxyribonucleotides with biological activity such as antisenseDNA (mRNA binding or triplex-forming DNA) or immunostimulatory CpGmotifs are characterized by sequence-specific linear motifs, oftenstabilized by intramolecular base-pair bonding. Backbone modification,such as phosphorothioate substitution, does not adversely affect andoften enhances the activity of these molecules.

[0010] We have previously described a composition and method comprising2 to 20 base 3′-OH, 5′-OH synthetic oligonucleotides selected from thegroup consisting of (G_(x)T_(y))_(n), (T_(y)G_(x))_(n),a(G_(x)T_(y))_(n), a(T_(y)G_(x))_(n), (G_(x)T_(y))_(n)b,(T_(y)G_(x))_(n)b, a(G_(x)T_(y))_(n)b, a(T_(y)G_(x))_(n)b, wherein x andy is an integer between 1 and 7, n is an integer between 1 and 12, a andb are one or more As, Cs, Gs or Ts, wherein the sequence is between 2and 20 bases and wherein the sequence induces a response selected fromthe group consisting of induction of cell cycle arrest, inhibition ofproliferation, induction of caspase activation and induction ofapoptosis in a number of cancer cells (PCT CA00/01467, WO 01/44465).

[0011] Computational procedures allow a correlation of three dimensionalmolecular structures with biological activity, and facilitate predictionof the conformation of active molecules. Modeling entails the use ofmathematical equations that are capable of representing accurately thephenomenon under study. Molecular mechanics analysis (Allinger, N. L.,J. Comput. Chem., 12, 844,1991) can be used to analyze structural andconformational relationships. The fundamental assumption of molecularmechanics is that data determined experimentally for small molecules(bond length, bond angles, etc.) can be extrapolated to largermolecules. Molecular modeling approaches have been used to determinestructure activity relationships and to enable the prediction of activethree dimensional molecular conformations (N. Evrard-Todeschi et al., J.Chem. Inf. Comput. Sci., 38:742,1998; Chen H. et al., J. Med. Chem.36:4094, 1993; A. Guama et al., J. Med. Chem., 40:3466,1997; M. Read, etal. Proc. Natl. Acad. Sci. USA, 98:4844, 2001).

[0012] Therefore, there is a continuing need for the identification ofnovel 3-dimensional conformations or structural motifs inoligonucleotides that are useful in predicting their biologicalactivity, particularly with regard to their capability to inducecellular responses in cells. What is needed is the ability to predictcellular responses including responses such as apoptosis in cancercells.

SUMMARY OF THE INVENTION

[0013] The present invention fulfills this need by providing acomputer-based method useful for predicting whether oligonucleotidesequences possess apoptotic activity. This in silico method alsopredicts the relative efficacy of the oligonucleotide sequence to induceapoptosis. This invention provides a rational basis for in silicoevaluation or screening of oligonucleotide compositions for theirability to induce apoptosis, thereby providing a means to selectspecific oligonucleotide compositions for further testing in vivo or invitro. This invention provides significant savings in the cost of drugdesign and development by: a) identifying oligonucleotide compositionswith specific predicted biological activity; b) predicting the efficacyof the oligonucleotide compositions with the specific predictedbiological activity; and, c) reducing the number of candidateoligonucleotide compositions to be tested in vitro and in vivo forapoptotic activity.

[0014] Prediction of the capability of a sequence to induce apoptosis isdesired in several diseases and conditions including but not limited tothe following: cancer; hyperproliferative disorders; autoimmune disease;arthritis; rheumatoid arthritis; inflammation; lymphoproliferativedisorders; restenosis of vessels after angioplasty; and, asthma.Prediction of the capability of a sequence to induce apoptosis isparticularly desirable in cancers including but not limited to, squamouscell carcinoma, fibrosarcoma, sarcoid carcinoma, melanoma, mammarycancer, lung cancer, colorectal cancer, renal cancer, osteosarcoma,cutaneous melanoma, basal cell carcinoma, pancreatic cancer, bladdercancer, brain cancer, ovarian cancer, prostate cancer, leukemia,lymphoma and metastases derived therefrom.

[0015] The unexpected ability to predict apoptosis-inducing activity insilico with a high degree of precision (>95%) reduces the need forcostly high-throughput chemical synthesis and apoptosis-inducingscreening, thus enabling the identification of biologically activemolecules in a much more efficient and cost effective manner.

[0016] An advantage of the present invention is that it accelerates thediscovery of new therapeutic compositions. Another advantage of thepresent invention is that it decreases the cost of discovering newtherapeutic compositions by providing candidate oligonucleotidesequences for biological testing in vivo and in vitro. These savingsdirectly affect the cost of therapeutic drugs for patients andthroughout the health care industry for humans and animals. Stillanother advantage of the present invention is that it decreases the costof discovering new therapeutic compositions by predicting the efficacyof oligonucleotide sequences, thereby providing a prioritization forbiological testing in vivo and in vitro.

[0017] Accordingly, it is an object of the present invention is toprovide a computer-based method useful for evaluation of oligonucleotidesequences.

[0018] It is another object of the present invention to provide acomputer-based method useful for evaluation of oligonucleotide sequencesto predict whether they possess the ability to induce a response in acell such as inhibition of cellular proliferation, induction of cellcycle arrest or induction of apoptosis.

[0019] It is a specific object of the present invention to provide acomputer-based method useful for evaluation of oligonucleotide sequencesto predict whether they possess the ability to induce apoptosis incells.

[0020] Yet another object of the present invention is to provide acomputer-based method useful for evaluation of oligonucleotide sequencesto predict whether they possess the ability to induce apoptosis incancer cells.

[0021] Yet another object to the present invention is to provide amethod useful for identifying oligonucleotide sequences that will beuseful in the treatment of disease.

[0022] Another object to the present invention is to provide a methoduseful for identifying oligonucleotide sequences that will be useful inthe treatment of diseases and conditions characterized by undesiredcellular proliferation.

[0023] Still another object to the present invention is to provide amethod useful for identifying oligonucleotide sequences that will beuseful in the treatment of diseases and conditions characterized byundesired cellular proliferation such as autoimmune disease,inflammation, arthritis, asthma, restenosis of vessels afterangioplasty, hyperproliferative disorders, lymphoproliferative disease,and cancer.

[0024] Yet another object to the present invention is to provide amethod useful for identifying oligonucleotide sequences that will beuseful in the treatment of cancer.

[0025] Another object to the present invention is to provide a methodthat allows the identification of molecules with apoptosis-inducingactivity in silico without resort to high throughput chemical synthesisand biological activity screening.

[0026] The unexpected and surprising ability of the present invention topredict the capability and efficacy of an oligonucleotide sequences toinduce a cellular response, and particularly to inhibit cellproliferation, to arrest the cell cycle progression and/or to induceapoptosis in cells addresses a long unfulfilled need in the medical artsand provides an important benefit for animals and humans.

[0027] These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiment and the appended claims.

BRIEF DESCRIPTION OF THE FIGURES

[0028] The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawings will be provided by the Office uponrequest and payment of the necessary fee.

[0029]FIG. 1. Spatial definition of the general orientation of a centrumin three dimensions (front, rear, ventral, dorsal, lateral left andlateral right.

[0030]FIG. 2a. Schematic representation of framed centrum ofelectronegativity in three dimensions.

[0031]FIG. 2b. Schematic representation of framed centrum ofelectronegativity in three dimensions showing x, y, z, alpha (α) andbeta (β) variables as defined in the specification and the 5′ and 3′orientation. Also shown are areas of electronegativity from phosphategroups, areas of electropositivity from amines and amido groups,phosphates, deoxyribose, and bases.

[0032] FIGS. 3-13. The following FIGS. 3-13 each consist of views asfollows:

[0033] a) Position 1 as starting position

[0034] b) Position 2 after 90° rotation along the x-axis from thestarting position

[0035] c) Position 3 after 180° rotation along the x-axis from thestarting position

[0036] d) Position 4 after 270° rotation along the x-axis from thestarting position

[0037] e) Position 1 as starting position in black and white showing thesolvent accessible surface.

[0038] The first position is presented twice as a solid, black and whitepicture and as a color picture with dots as the solvent accessiblesurface. The hydrogen atoms are suppressed for better clarity. Thefollowing colors are used to represent the atoms in the 3-dimensionalsequences shown in the figures: Blue=nitrogen; Grey=carbon;Pink=phosphorus; Red=oxygen; Yellow=sulfur. The four rotational views(a, b, c, and d) are provided as examples to demonstrate the globularnature of the centrum in different orientations. FIG. 3. TGT FIG. 4.GGGGGG FIG. 5. GGGTGG phosphorothioate backbone FIG. 6. GGG FIG. 7.TTGTGG FIG. 8. GGGTGGGG FIG. 9. GGGTGG_3P (3′-phosphate) FIG. 10.5P_GGGAGG (5′-phosphate) FIG. 11. 5P_GGGTGG (5′-phosphate) FIG. 12.GGGTGG FIG. 13. GGGGTGG

DETAILED DESCRIPTION OF THE INVENTION

[0039] The present invention provides a computer-based method useful forpredicting whether oligonucleotide sequences possess the ability toinduce a cellular response, and particularly to inhibit cellproliferation, to arrest the cell cycle progression and/or to induceapoptosis in cells. In a preferred embodiment, this in silico methodpredicts whether oligonucleotide sequences possess the ability to induceapoptosis. This in silico method also predicts the relative efficacy ofthe oligonucleotide sequence to induce a cellular response, andparticularly to inhibit cell proliferation, to arrest the cell cycleprogression and/or to induce apoptosis in cells. In a preferredembodiment, this in silico method predicts the relative efficacy of theoligonucleotide sequence to induce apoptosis in cells. This inventionprovides a rational basis for in silico evaluation or screening ofoligonucleotide compositions to predict their ability to inhibit cellproliferation, to arrest the cell cycle progression, to activatecaspases, to cleave PARP, and/or to induce apoptosis in cells, therebyproviding a means to select specific oligonucleotide compositions forfurther testing in vivo or in vitro.

[0040] The method of the present invention is used to determine whetheran oligonucleotide sequence possesses one or more centrums which areuseful in predicting whether the sequence has apoptotic biologicalactivity.

[0041] As used herein, sequence refers to an association of bases,deoxyribose and phosphodiester groups in an oligonucleotide sequenceforming an identifiable globular 3-dimensional structure (that is basedon the centrum of negatively charged phosphate groups framed by positivecharges of amino/amido groups of bases at the opposite side) that isused to predict the capability of the sequence to induce apoptosis incells.

[0042] As used herein, “centrum” refers to the absence or presence ofintramolecular substructure comprising two or more phosphate groups andtwo or more adjacent bases (Type A centrum) or non-adjacent bases (TypeB centrum), with or without stabilizing intermolecular hydrogen bonding.The centrum is defined as adjacent (type A) if bases are adjacent with aperpendicular orientation in the same or opposite plane to the phosphatenecklace. The centrum is defined as non-adjacent (type B) if the orderof the bases is not consecutive. A preferred orientation is type A or B,a more preferred orientation is type A and B, a most preferredorientation is type A with the bases in the same plane perpendicular tothe phosphate necklace. If a sequence has one centrum at the 5′ end anda second centrum at the 3′ end then the subscript index, e.g. A₁ refersto the type A centrum at the 5′ end. The subscript A₂ refers to the typeA centrum at the 3′ end. The same indexing is applied to the type Bcentrum. The centrum is considered as framed if there is presence ofamino or amido or both groups at the opposite sites of the phosphatenecklace.

[0043] The following notation is used to describe the sequence of basesin the oligonucleotide sequences: A=Adenine; C=Cytosine; G=Guanine;T=Thymine.

[0044] The following parameters are used in describing the 3-dimensionaloligonucleotide sequences: A) All distances are in pm; B) Intramolecularhydrogen bonds are assumed to form if the mutual distance ofparticipating atoms is less than 300 pm; and, C) The molecular dynamicsvalues are in kcal/mole.

[0045] As used herein, a cellular response refers generally toinhibition of proliferation, to arrest in cell cycle progression and/orto induction of apoptosis in cells. A preferred response is induction ofapoptosis. Cells include any cell, particularly cells exhibitingundesired proliferation. Such cells may be found in hyperproliferativedisorders; autoimmune disease; arthritis; rheumatoid arthritis;inflammation; lymphoproliferative disorders; cancer; and, asthma. Cancercells include, but are not limited to, cells from squamous cellcarcinoma, fibrosarcoma, sarcoid carcinoma, melanoma, mammary cancer,lung cancer, colorectal cancer, renal cancer, osteosarcoma, cutaneousmelanoma, basal cell carcinoma, pancreatic cancer, bladder cancer, braincancer, ovarian cancer, prostate cancer, leukemia, or lymphoma andmetastases derived therefrom.

[0046] As used herein, the phrases “therapeutic treatment” and “amounteffective to” refer to an amount of a 3-dimensional oligonucleotidesequence effective to inhibit cell proliferation, arrest the cell cycleprogression or induce apoptosis in cells, including cancer cells.

[0047] Administration of a composition comprising an effective amount ofa oligonucleotide sequence of the present invention to an animal,including a human, is a therapeutic treatment that prevents, treats oreliminates a disease, including, but not limited to, cancer, arthritis,rheumatoid arthritis, hyperproliferative disorders, restenosis ofvessels after angioplasty, lymphoproliferative disorders and asthma.

[0048] Induction of apoptosis is particularly desirable in cancersincluding but not limited to, squamous cell carcinoma, fibrosarcoma,sarcoid carcinoma, melanoma, mammary cancer, lung cancer, colorectalcancer, renal cancer, osteosarcoma, cutaneous melanoma, basal cellcarcinoma, pancreatic cancer, bladder cancer, brain cancer, ovariancancer, prostate cancer, leukemia, lymphoma and metastases derivedtherefrom.

[0049] Although not wanting to be bound by the following statement, itis proposed that a molecular combination in an oligonucleotide sequenceof negative charges or electronegativity (from for example phosphategroups), positive charges or electropositivity (from for example amineand amido groups), in conjunction with appropriate intra-molecularhydrogen bonding and inter-atomic dimensions as defined herein, willpossess the ability to induce apoptosis in cancer cells.

[0050] Computer Hardware and Software

[0051] It is to be understood that the present invention may bepracticed using any computer with sufficient memory and computing speedto operate chemical drawing software used by those of ordinary skill inthe art and to measure the parameters of the centrum. Measurements ofthe various parameters of the centrum are accomplished by means of thesoftware translates drawn structures into 3-d structures.

[0052] Using the software, interatomic distances are measured in pm(picometers) in the 3-dimensional structure where the first atom isselected and at the tip of the cursor (pointer) the information aboutthe distance between those atoms is shown.

[0053] Further, any chemical software that facilitates determination ofthe components of the centrum described below may be used. Such softwareis commonly known to those of ordinary skill in the art. In oneembodiment of the present invention, ChemDraw software is employed. TheChem3D software is supplied by Cambridgesoft.Com, Cambridge, Mass., USA.Other software packages known to one of ordinary skill in the art ofmolecular modeling may be employed, such as Sybill (from Tripos), Charmand Inside II (from Accelrys).

[0054] The method of the present invention may be practiced usingpersonal computers such as commonly available to consumers, for example,desktop units and laptops manufactured by Dell, Apple, Compaq,Hewlett-Packard, Gateway, IBM or more sophisticated computers such asSilicon Graphics or Cray computers.

[0055] The computer is operationally connected to a means for entry ofinformation, such as a keyboard, touchscreen or other entry device knownto one of skill in the art. The computer is operationally connected tomeans such as CD read write devices, disk drive or other means known toone of skill in the art for accessing and inputting information.Further, computer may be operationally connected to the internet, toremote databases, or to other servers that provide access to databasesof chemical structures so that information concerning specific molecularstructures may be obtained rapidly.

[0056] The computer is operationally connected to a means for display oroutput of information, such as monitors, printers and other displaymeans known to one of skill in the art. Such means permit visualizationof three dimensional structure of oligonucleotide sequences and variousparameters associated with their structure, such as globular shape,shape of the phosphate backbone orientation and location of phosphategroups, 2-deoxyribose, purines, pyrimidines, amino groups, amido groups,hydroxyl groups at 3′ and 5′ ends, and centrums.

[0057] General Method of the Invention for in Silico Identification ofCentra in Oligonucleotides

[0058] The following steps describe the general method of the presentinvention for identifying centra in an oligonucleotide sequence.

[0059] 1) Draw the molecule. The oligonucleotide molecule was drawnusing ChemDraw v.5 software

[0060] 2) Check for errors Structures were examined to ensure that theatoms, bonds and valences were correct.

[0061] 3) Translation and analysis of drawn model by Chem3D software.The structure was drawn and then opened using the Chem3D softwarestructure to create a 3-dimensional model. If an error was found (e.g.,double bonds instead of single bonds) a warning message was generatedindicating that something was not correct. In such case the ChemDrawprogram was used to make changes in the drawn structure, and theprocedure was repeated by opening the drawn (corrected) structure inChem3D software.

[0062] 4) Minimization of energy. Minimization of energy was requiredfor locating stable conformations. From the MM2 menu in CHem3D software,the “Minimize energy” choice was made. The default value of 0.1000 was areasonable compromise between accuracy and speed. The result containedthe values of the following parameters: bond stretching energy; anglebending energy; torsional energy; non-bonded energy; van der Waalsenergy; electrostatic energy; dipole/dipole contribution; dipole/chargecontribution; out-of-plane bending; and, stretch-bend parameters. Thestretch-bend parameters are force constants for the stretch-bendinteraction terms in the prior list of elements. Parameters are alreadyinstalled as a part of the software. X and y represent any non-hydrogenatom. when an angle is compressed, the MM2 force field uses thestretch-bend force constants to lengthen the bonds from the central atomin the angle to the other two atoms in the angle.

[0063] The Total Steric Energy for the given conformation is expressed asummary of the values mentioned above (bond stretching energy, anglebending energy, torsion energy, van der Waals energy, electrostaticenergy and stretch-bend energy) in units of kcal/mol. Stretch bend crossterms are used when a coupling occurs between bond stretching and anglebending. The sum of these energies gives the resulting total stericenergy.

[0064] 5) Calculation of molecular dynamics of molecule at 310° KMolecular Dynamics calculations used Newtonian mechanics to simulatemotion of atoms, adding or subtracting kinetic energy as the model movesfrom lower to higher temperature or vice versa.

[0065] The Molecular Dynamics was computed from the Menu MM2 by choosingMolecular Dynamics. The present computation used the default parametersas follows: step interval: 2fs; frame interval: 10 femtosecond (fs);terminate after: 10,000 steps; heating/cooling rate: 1 kcal/atom/1picosecond (ps); target temperature set at 300° Kelvin (K).

[0066] The target temperature was set at 300° K after a set ofexperiments to determine variance between 300° K and 310° K with respectto the shape of molecules and the temperature range of each molecule. Itwas found that the default value of 300° K covered the range of thetemperature up to 310° K (300° K corresponded to the temperature of 37°C. at which the experiments were performed). It was observed that if thetemperature was set to 310° K the range of calculated values usuallyexceeded the 310° K range.

[0067] 6) Display of solvent accessible surface to identify molecularconformation, base fingers and necks between them, and to identifyglobular or linear domains of given molecule. Solvent accessible surfacedisplays provide information about entire molecules instead of specificatom and bond information. The solvent accessible surface represents theportion of the molecule that solvent molecules can access. Commonsolvents have different values of radius. The default value of water(140 pm) was used. Surfaces display information about the molecule'sphysical and chemical properties. Surfaces display aspects of theexternal surface interface (or electron distribution) of a molecule.Molecular surface types are solid, wire mesh, dots, or translucent. Todisplay the molecular surfaces the View menu was employed with fourchoices: A) solid the surface is displayed as an opaque form. This wasuseful to examine details of the surface itself and not particularly inthe underlying atoms and bonds. B) Wire mesh was displayed as aconnected net of lines. The wire mesh displays surface features andpermits visibility of the atoms and bonds. C) Dots were displayed as aseries of unconnected dots. This was we used to view the underlyingstructure. D) Translucent surface is displayed in solid form but ispartially transparent so that the atoms and bonds are visible.

[0068] 7) Identification of intramolecular hydrogen bonds. Hydrogenbonds are capable of being formed if the distance is equal to or lessthan 300 pm.

[0069] 8) Identification of the presence of phosphate groups forming aphosphate necklace or bead-like appearance as the basis for theformation of a strong electronegative centrum. If a phosphate necklacewas present, the size of the centrum was calculated.

[0070] 9) Determination of the spatial orientation of bases forelectropositive framing with respect to phosphate groups in anelectronegative centrum.

[0071] 10) Measurement of interatomic distances of amino/amido groups,and 3′ and 5′ hydroxyl groups from phosphate groups. Using the software,interatomic distances were measured in pm (picometers) in the3-dimensional structure where the first atom is selected and at the tipof the cursor (pointer) the information about the distance between thoseatoms is shown. Interatomic distances: The relative position of eachatom in our models was determined by a set of measurements calledinternal coordinates of Z-matrix. The internal coordinates for anyparticular atom consists of measurements, in the present case bondlength between it and other atoms (more detailed analyses optionallyinclude bond angles and dihedral angles).

[0072] Internal coordinate values were obtained by choosing values fromthe tools menu, pointing to show model tables, and then choosinginternal coordinates). The first three atoms in a Z-matrix were definedas follows: 1) the origin atom was the first atom in Z-matrix, and allother atoms in the model were positioned in terms of this atom; 2) thefirst positioned atom was positioned only in terms of the origin atom.The first positioned atom position was specified by the distance fromthe origin atom; (in the present case it was the measurement ofinteratomic distances between phosphate groups, between phosphate groupsand amino/amido groups, and between amino/amido groups of involvedbases); 3) The second positioned atom is positioned in terms of theorigin atom and the first positioned atom. the entire set of internalcoordinates was obtained from the tools menu by pointing to show modeltable and choosing internal coordinates.

[0073] 11) Comparison of model prediction with actual degree ofapoptosis-induction.

[0074] A comparison was made of the globular or linear shape andinteratomic distances of the electronegative centrum framed byamino/amido groups, and measured apoptotic activity.

[0075] 12) If two centra were found, then there is a high probability ofa higher degree of apoptosis-induction.

[0076] Description of Electronegative Centrum Framed by Amino/AmidoGroups of Bases as well as by Hydroxyl Groups at 5′ and 3′ ends.

[0077] The following description of a centrum includes its key features.FIGS. 2a and 2 b displays these features. FIG. 1 provides generalorientation of a centrum (front, rear, ventral, dorsal, lateral left andlateral right).

[0078] Orientation of bases in the centrum. The centrum is defined asadjacent (type A) if bases are adjacent with a perpendicular orientationin the same or opposite plane to the phosphate necklace. The centrum isdefined as non-adjacent (type B) if the order of the bases is notconsecutive. A preferred orientation is type A or B, a more preferredorientation is type A and B, a most preferred orientation is type A withthe bases in the same plane perpendicular to the phosphate necklace. onesequence can possess both type A and type B. If two centra were found inthe given sequence then A₁ represents the first centrum at the 5′ endand A₂ represents the second centrum at the 3′ end.

[0079] Centrum phosphorus atoms. the x-axis represents the inter-atomicdistance between phosphorus atoms associated with the centrum. If twophosphate atoms are involved (e.g. 5′-N_(1p)N_(2p)N₃-3′ where N_(X)represent purine or pyrimidine deoxyribonucleosides and P represents thephosphate group) then x is between approximately 700 to 1360 pm. If fourphosphate atoms are involved then x is between approximately 750 to 1300pm (e.g. 5′-N_(1p)N_(2p)N_(3p)N_(4p)N₅-3′ where N_(x) represent thenumber of purine or pyrimidine deoxyribonucleosides as an integer in therange 2 to 4, and P represents the number of phosphate groups as aninteger).

[0080] Phosphate backbone-base distance. The Y-axis represents thelongest distance between the phosphate backbone and the farthest atom ofa participating base. This will depend on the rotation of the given baseand which atom (group) is considered (methyl, carbonyl, or amino group).Y is equal to or between approximately 780 to 2200 pm. There is nopreferred distance for Y.

[0081] Centrum depth. The Z-axis represents the distance from the frontto the rear of the centrum when viewed from above. Z is equal to orbetween about 400 to 1300 pm. There is no preferred distance.

[0082] Amine/amido framing. The next defining parameter α is thefurthest framing distance of the amino/amido group from the phosphategroups. The α distance is equal to or between about 900 and 1500 pm.

[0083] Amine/amido distance. The next defining parameter β is thefurthest inter-atomic distance of amino/amido groups. The value is equalto or between about 300 to 1700 pm.

[0084] Intra-molecular hydrogen bonding. This bonding stabilizes thesize and shape of molecules as well as the interatomic distances.Hydrogen bond distances should be equal to or less than about 300 pm.The most frequently observed bonding is via carbonyl groups and amino oramido groups. The next most frequent type of hydrogen bonding is betweencarbonyl groups and hydroxyl groups at the 3′ or 5′ end of the molecule.The last type of hydrogen bonding is between carbonyl groups and thehydroxyl groups of phosphates. A preferred type of hydrogen bondingcomprises all three types, a more preferred hydrogen bonding is viacarbonyl groups and amino/amido groups, and via carbonyl groups andphosphate groups, and a most preferred hydrogen bonding is via carbonyland amino/amido groups.

[0085] Distance of the 5′ or 3′ end hydroxyls to their correspondingphosphate groups.

[0086] The last parameter is the distance of the 5′ or 3′ end hydroxylsto their corresponding phosphate groups. A preferred distance is equalto or between about 320 and 650 pm, a more preferred distance is equalto or between about 390 to 420 pm, and a most preferred distance isabout 380 pm.

[0087] Randomly oriented molecules have bases that form fingers withnecks separating individual fingers. These structures do not possess acentrum or centra as defined above and are either inactive or possessweak activity (<20% apoptosis-inducing activity, see Table 4).

[0088] The present invention demonstrates that a molecular combinationof negative charges or electronegativity (for example from phosphategroups), positive charges or electropositivity (for example from amineand amido groups), in conjunction with appropriate intra-molecularhydrogen bonding and with X, Y, Z, α and β dimensions as defined above,possesses the ability to induce apoptosis in cancer cells. Accordinglythe present invention provides a method of in silico analysis ofmolecular structures, particularly oligonucleotide structures, forprediction of apoptotic activity.

[0089] Three-dimensional computation is used to identify one or more3-dimensional centrum or centra in oligonucleotide sequences or othermolecules that comprise a molecular combination of negative charges orelectronegativity (from phosphate groups), positive charges orelectropositivity (from amine and amido groups), in conjunction withappropriate intra-molecular hydrogen bonding and with X, Y, Z, α and βdimensions as defined herein, and that on biological testing demonstratethe ability to induce apoptosis in cancer cells.

[0090] It is proposed that such a molecular combination of negativecharges or electronegativity (from for example phosphate groups),positive charges or electropositivity (from for example amine and amidogroups), in conjunction with appropriate intra-molecular hydrogenbonding and with X, Y, Z, α and β dimensions as defined above, willpossess the ability to induce apoptosis in cancer cells. While such amolecular combination is described for oligonucleotide sequences, it isclear that this approach can be used to conduct molecular modeling andidentification of apoptosis inducing centra in other molecular species.

[0091] In addition to the assay presented in Example 3 involving Annexinstaining, other in vitro assays may optionally be employed to evaluatecentrum-predicted biological activity of sequences. In vivo assays mayalso be employed. Various assays useful for this purpose are describedin PCT CA00/01467, WO 01/44465, the entirety of which is incorporatedherein by reference. Additional assays for evaluation of the efficacy ofthe sequences are described by Oncogene Research Products, P.O. Box12087, La Jolla, Calif. 92039 (Apoptosis Catalog and Technical Guide2002-2003, especially pages 5-295) the entirety of which is incorporatedherein by reference. Such assays include assays designed to analyze DNAfragmentation, apoptosis, mitochondrial markers, endoplasmic reticulummarkers, free nucleosomes, nuclear matrix proteins, detection andactivity of numerous caspases and related proteins, including but notlimited to caspases 1 through 14, glutathione, superoxide dismutase,members of the bcl-2 family, analysis of the Fas/TNR-R super family,PARP related products, analysis of apoptotic signal transducers,analysis of various signaling receptors including death receptors, Apo2,decoy receptors, analysis of apoptotic membrane proteins, nervous systemapoptotic markers, numerous markers for cell cycle and cellularproliferation, mitotic kinases, bromodeoxyuridine assays, and p53assays.. The evaluation of the efficacy of the sequences identified withthe analytical method of the present invention may also be determined inthe presence of other agents, and therapeutic agents, such as inducersof apoptosis and cell synchronization reagents as described by OncogeneResearch Products, P.O. Box 12087, La Jolla, Calif., 92039 (ApoptosisCatalog and Technical Guide 2002-2003, especially pages 99-104 and pages214-255, the entirety of which is incorporated herein by reference).Such agents include but are not limited to actinomycin D, amphidocolin,A23187, caffeine, camptothecin, cycloheximide, dexamethasone,doxorubicin, 5-fluorouracil, hydroxyurea, paclitaxel, staurosporine,thymidine, vinblastine, retinoic acid, etoposide, okadaic acid,vincristine and methotrexate.

[0092] The following examples will serve to further illustrate thepresent invention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various embodiments, modifications and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the invention.

EXAMPLE 1

[0093] Preparation of Deoxyribonucleic Acid Sequences

[0094] Phosphodiester and phosphorothioate sequences were prepared bySigma-Genosys (Woodlands, Tex.) using Abacus Segmented SynthesisTechnology. Unless stated otherwise, the sequences were dispersed inautoclaved deionized water or in a pharmaceutically acceptable buffersuch as, but to limited to, saline immediately prior to use.

EXAMPLE 2

[0095] Cells and Treatment

[0096] Human Jurkat T cell leukemia cells were obtained from theAmerican Type Culture Collection (Rockville, Md.). The Jurkat T cellswere maintained RPMI 1640 medium, supplemented with 10% heat-inactivated(56° C., 30 min) fetal bovine serum (all from Sigma Aldrich, Canada) inan atmosphere of 5% CO₂ at 37° C. Cells were seeded at 2×10⁵ cells/mlmedium in 6-well float-bottomed tissue culture plates and incubated witholigonucleotides at a final concentration of 53 μM. Testing of otherconcentrations of the oligonucleotides demonstrated that they inducedapoptosis in a concentration dependent manner.

EXAMPLE 3

[0097] Analysis of Apoptosis

[0098] Apoptosis was measured by staining cells with Annexin V FITC andpropidium iodide (PI) (BD Pharmingen, San Diego, Calif. USA) accordingto the manufacturer's instructions. Flow Cytometry (FCM) determinedcellular fluorescence. The FCM and data analysis were carried out usinga FACSCalibur instrument (excitation 488 nm, emission 530 nm forAnnexin-V and 580 nm for PI) using the program CELLQuest.

EXAMPLE 4

[0099] 3 Dimensional Molecular Modeling

[0100] Chem3D version 5.0 and 6.0 software (CabridgeSoft Corporation,Cambridge, Mass.) was used to create 3-dimensional images of specificsequences. Molecular mechanics computation of minimal energyconformations (MM2; Allinger N. L., J. Comput. Chem., 1993, 14:755-68)was carried out at a default value 300° K using Newtonian mechanics tosimulate motion of atoms, adding kinetic energy as the model'stemperature increased. The values of molecular dynamics as well as thetemperature range in which the molecular dynamics is valid arementioned. 3-dimensional modeling was carried out in order to identifythe absence or presence of intramolecular grouping, defined as acentrum. The spatial arrangement of electronegative charges (phosphateand base carbonyl groups, and electropositive charges (amino, amido, orhydroxyl groups at 3′ and 5′ ends) were also analyzed. Whenintramolecular grouping was observed in the 3-dimensional models,spatial characteristics were defined according to localization ofphosphate groups, localization of amine/amido groups, and position ofhydroxyl groups, or intramolecular grouping(s) in the oligonucleotides.The resulting structures are presented with the 5′-end at the left and3′-end at the right.

[0101] The spatial orientation is characterized as shown in FIG. 1.Although not wanting to be bound by the following hypothesis, it isthought that the simplified ideal 3-dimensional sequence shown in FIG.2a consists of phosphate groups (circles) and 2-deoxyribose units(cylinders) at the ventral position and horizontally oriented bases(prisms) at the dorsal side.

EXAMPLE 5

[0102] ODN with Relatively Weak (<20%) Apoptotic Activity

[0103] The results of the computational analysis and correlation withapoptosis-inducing activity are summarized in Table 1. Oligonucleotidescontaining between 3 and 8 bases and apoptosis values of less than 20%do not possess an identifiable 3-dimensional framed centrum. Typicalillustrative 3-dimensional structures of sequences with weak apoptosisactivity are shown in FIGS. 3, 4, and 5. TABLE 1 Date of sequence withweak (<20%) apoptotic activity Number Framed of bases Sequence Apoptosis(%) centrum H bonds 3 TGT 13 0 No 6 CCGTCC 5 0 Yes CTGTCT 14 0 YesGGGCGG 17 0 Yes GGGGGG 4 0 No TCGTTC 9.5 0 Yes GGGTGG 0 0 Yesphosphorothioate 7 GGGGGTG 11 0 No 8 GGGGGTGG 19 0 Yes

EXAMPLE 6

[0104] ODN with Intermediate (>20%<40%) Apoptotic Activity.

[0105] The results of the computational analysis and correlation withapoptosis-inducing activity are summarized in Table 2. Oligonucleotidescontaining between 3 and 8 bases and apoptosis activity of >20%<40%possessed an identifiable centrum of either type A or type B. Typicalillustrative 3-dimensional structures are shown in FIGS. 6-8. TABLE 2Data of sequences with intermediate apoptotic activity P number/ # ofFramed Centrum number of Alpha Beta bases Sequence % apoptosis centrumtype bases X(pm) Y(pm) Z(pm) (pm) (pm) H bond 3 GTG 27 1 A 3/2 721 1252759 1150 994 Yes 4 GTGG 23 1 A 4/3 1262 1219 863 1125 936 Yes 5 GGTGG 211 A 3/4 1202 1403 882 1207 757 Yes 6 AAGTAA 23 1 A 2/2 728 1064 672 916751 No ATGTAT 37 1 A 2/2 748 998 599 934 428 Yes GGCCGG 21 1 B 2/4 7761093 882 1414 731 Yes TTGTGG 38 1 A 2/2 737 783 553 970 726 Yes GGGTGGG29 1 A 3/3 702 1149 893 982 853 Yes 8 GGGTGGGG 23 1 A 2/2 777 1342 848948 1157 Yes

EXAMPLE 7

[0106] ODN with High (≧41%≦80% Apoptosis) Activity

[0107] The results of the computational analysis and correlation withapoptosis-inducing activity are summarized in Table 3. Oligonucleotidescontaining between 3 and 7 bases and apoptosis activity of ≧41%≦80%possessed an identifiable centrum of either type A or type B. Typicalillustrative 3-dimensional structures are shown in FIGS. 9-13.

EXAMPLE 8

[0108] Influence of Phosphodiester Versus Phosphorothioate Backbone On3-Dimensional Conformation and Apoptotic Activity

[0109] Comparison of the activity and 3-dimensional conformation ofsequence GGGTGG with a phosphodiester backbone (Table 3 and FIG. 12) andGGGTGG with a phosphorothioate backbone (Table 1 and FIG. 5) shows thatthe change in apoptosis-inducing activity (50% and 0% respectively)correlates with a loss of a framed centrum of strong electronegativitydue to the prevailing planar orientation of the first three Gs, as wellas the amino group of G₄ at the frontal bottom and the one of G₅ at thedorsal top excludes the last two Gs from the framed centrum. TABLE 3Data of sequences with high apoptotic activity P number/ # of FramedCentrum number of Alpha bases Sequence % apoptosis centrum type basesX(pm) Y(pm) Z(pm) (pm) beta(pm) H bond 4 GGTG 42 1 B 3/3 1123 1393 8981118 1257 Yes 5 GGGTG 46 1 A 2/2 724 1159 652 1049 818 Yes GTGGG 46 1 A2/3 658 1152 411 1180 826 Yes 6 GGGTGG_3P 46 1 A 3/3 1423 3132 1566 1174967 Yes 5P_GGGTGG 62 2 A₁ 3/3 1250 1125 845 1719 1793 Yes 5P_GGGTGG A₂2/2 747 1085 514 821 609 Yes GGAAGG 59 1 B 3/4 901 1262 759 1451 1396Yes GGTTGG 66 1 A 2/2 748 947 698 881 306 Yes GGGTGG 50 1 A 2/2 753 1149674 1101 613 Yes GTGGTG 43 1 A 3/4 1440 2197 1288 1191 1333 Yes GTGTGT67 1 A 3/3 1038 1686 1232 1222 1471 Yes TGGTTG 69 2 A₁ 2/2 788 1054 9491007 631 Yes TGGTTG A₂ 2/2 725 881 725 937 514 Yes TGTGTG 66 1 A 3/3 8871527 1231 1522 1086 Yes 7 GGGGTGG 44 1 A 2/2 702 1149 893 982 853 Yes

EXAMPLE 9

[0110] 3′ or 5′ Modification Does Not Result in the Loss ofApoptosis-Inducing Centra

[0111] 3′ modified sequences GGGTGG-phosphate (Table 3, FIG. 9),5′-modified sequence phosphate-GGGTGG (Table 3, FIG. 11) all contained aframed centrum. 3′, 5′ or 3′, 5′-modification of oligonucleotidescontaining a centrum of activity does not result in conformationalchanges that lead to the loss of such centra.

EXAMPLE 10

[0112] Prediction of the Apoptotic Efficacy of a Sequence

[0113] The sequences in Table 4 were analyzed with the method of thepresent invention to determine if they possessed a centrum. A predictionwas made as to whether the sequences would possess the ability to induceapoptosis, arbitrarily set at 20%. In other words, a prediction ofapoptotic activity implied activity greater than 20%. Subsequently, thesequences were tested in vitro for apoptotic activity. The results areshown in Table 4 and indicate a very high success rate in predictingapoptotic activity. The method was successful for each sequenceanalyzed. These data demonstrate that the in silico method of thepresent invention identifies sequences with different degrees ofapoptotic activity, thereby providing a basis for prioritizing selectionof sequences for biological testing. TABLE 4 Predictive value of themethod using new sequences with unknown apoptosis-inducing activityPRESENCE PREDICTED ACTUAL % OF A APOPTOTIC OF CELLS CENTRUM ACTIVITY INSEQUENCE YES NO No Yes APOPTOSIS GCG-(3 bases) X X 25 GGAG-(4 bases) X X35 AGTA-(4 bases) X X 12 GAGG-(4 bases) X X 17 GGAG-(4 bases) X X 35GAGGG-(5 bases) X X 66 GGGAG-(5 bases) X X 73 GGGGAG-(6 bases) X X 33GAGGGG-(6 bases) X X 13 GAGGGGG-(7 bases) X X 5 GGGAGGGG-(8 bases) X X76 GGGGGAGG-(8 bases) X X 11 AAAGTAAA-(8 bases) X X 6

[0114] It is to be understood that the foregoing relates only to apreferred embodiment of the present invention and that numerousmodifications or alterations may be made therein without departing fromthe spirit and scope of the invention as set forth in the appendedclaims.

We claim:
 1. A computer-based method of predicting biological activityof an oligonucleotide sequence comprising analysis of theoligonucleotide sequence to determine if the oligonucleotide sequencecontains at least one centrum.
 2. The method of claim 1, wherein theanalysis comprises examining the sequence for electronegativity,electropositivity, intra-molecular hydrogen bonding and inter-atomicdistances.
 3. A computer-based method of predicting biological activityof an oligonucleotide sequence comprising: drawing the sequence usingchemical drawing software; checking the drawn sequence for errors;creating a three dimensional model of the drawn sequence; minimizingenergy of the three dimensional model; calculating molecular dynamicsusing chemical analytical software; displaying a solvent accessiblesurface of the three dimensional model on a display means; identifyingglobular and linear domains in the three dimensional model; identifyingintramolecular hydrogen bonds; identifying phosphate groups capable offorming an electronegative centrum; evaluating spatial orientation ofbases in the three dimensional model for electropositive framing withrespect to the phosphate groups in the electronegative centrum;measuring interatomic distances of amino/amido groups, and 3′ and 5′hydroxyls from phosphate groups; and, predicting whether the sequencepossesses the biological activity.
 4. The method of claim 3, whereinmeasuring interatomic distances of amino/amido groups and 3′ and 5′hydroxyls from phosphate groups comprises: measuring a first interatomicdistance X between phosphates associated with the centrum; measuring asecond interatomic distance alpha between phosphates and a farthest atomin a participating base; measuring a third interatomic distance beta asthe furthest inter-atomic distance beta between amido or amino groups;measuring a fourth interatomic distance Z between a front to a rear ofthe centrum; and, measuring a fifth interatomic distance Y as a longestdistance between a phosphate backbone and the farthest atom in aparticipating base.
 5. The method of claim 4, wherein a centrum ispresent in the sequence if Y is equal to or between about 780 to 2200pm; Z is equal to or between about 400 to 1300 pm; alpha is equal to orbetween about 900 and 1500 pm; beta is equal to or between about 300 to1700 pm; and, X is between about 700 to 1360 pm.
 6. The method of claim1, wherein the biological activity is inhibition of cellularproliferation, induction of cell cycle arrest or induction of apoptosis.7. The method of claim 3, wherein the biological activity is inhibitionof cellular proliferation, induction of cell cycle arrest or inductionof apoptosis.
 8. The method of claim 1, further comprising testing thesequence for the biological activity.
 9. The method of claim 3, furthercomprising testing the sequence for the biological activity.